Figure 3

Treatment of isolated splenocytes with 5-Aza increases the expression of Foxp3 and GFP. (A) Isolated murine splenocytes were activated by anti-CD3 (1 µg/mL), anti-CD28 (0.2 µg/mL) and mouse IL-2 (10 ng/mL). Cells were treated with 1 µmol/L 5-Aza (solid circles) or DMSO alone (open circles) every day for 4 d. Foxp3 mRNA expression was measured by quantitative real-time PCR with normalization to β-actin (**p < 0.001). (B, C) CD3⁺GFP⁻ T cells were harvested from Foxp3-EGFP C57BL/6 transgenic mice (C.Cg-FOXP3^tm2Tch/J) and isolated by a BD FACSAria III cell sorter. The isolated CD3⁺GFP⁻ T cells were activated as above and then treated with 1 µmol/L 5-Aza or DMSO alone. Flow cytometry was used to monitor GFP expression. A representative flow cytometry result is shown (B). Results are summarized from d 0 to d 4, with 5-Aza (solid circles) and DMSO alone (open circles) (C) (*p < 0.05). All data are representative of three independent experiments with triplicate culture wells.